The focus of these studies is on carboxypeptidase E (CPE). This enzyme is thought to be an essential enzyme involved in the production of most opiate peptides (such as enkephalin, dynorphin, and related peptides), as well as numerous peptides in brain and other tissues. Studies on CPE are important to further our knowledge about this key enzyme, and to provide a useful model system for other neuropeptide processing enzymes. During the past 12 years since CPE was first identified, the cDNA and gene have been cloned, the regulation of CPE has been examined in a wide variety of systems, and enzymes which process the CPB precursor, proCPE, have begun to be characterized. Over the next five years, we will focus on three related areas that address important questions in the field of neuropeptide processing enzymes. 1) To define the region of CPE involved with membrane binding and sorting into the regulated secretory pathway. We will test the hypotheses that (i) the C-terminal region of CPB is both a membrane anchor and a sorting signal for entry into the regulated secretory pathway; and (ii) Ca2+ induced aggregation of CPE contributes to the sorting of this protein into the regulated secretory pathway. These studies will provide a better understanding of protein sorting into the regulated pathway by testing current models for this sorting. 2) To eliminate CPE from a cell line by gene disruption. These experiments will test the hypothesis that CPE is the only carboxypeptidase involved in neuropeptide biosynthesis. While there is considerable evidence for this hypothesis (discussed in Background and Significance), final proof requires deleting CPB and showing that peptide processing is not complete. These experiments will also reveal whether CPE performs additional functions in the cell, or if any other carboxypeptidase activities participate in neuropeptide production; this may lead to the identification of novel carboxypeptidases. 3) To characterize secretory vesicle enzymes which process proCPE. We have found enzymes in secretory granules which process proCPE into CPE, and cleave the membrane-bound form into the soluble form. These enzymes are distinct from the well characterized neuropeptide processing enzymes. We will purify and characterize these novel enzymes. Since it is likely that the proCPE processing enzymes perform other roles within the cell, these studies should be of broad relevance. For example, the proCPE-cleaving enzyme may also process secretory granule proteins (such as other peptide processing enzymes) or viral proteins (such as the human immunodeficiency virus envelope glycoprotein gp160). Together, these complementary studies will provide important information on CPE, a key enzyme in the production of enkephalins and other molecules that are relevant to the biochemistry of drug abuse.